Study On The Paracrine Effect Of Bone Morphogenetic Proteins(BMP7) During Ovarian Development Of The Mud Crab,Scylla Paramamosain

In vertebrates,bone morphogenetic proteins(BMPs)play an important role in various biological processes,such as bone formation,growth,differentiation and reproduction.BMPs have an important impact on reproduction through the BMPs-SMADs(drosophila mothers against decapentaplegic protein)signaling pathway.BMPs are secreted from oocytes or follicle cells,and binding to two types of receptors(BMPRI and BMPRII)in oocytes or follicle cells via an autocrine/paracrine manner.Subsequently,BMPRI phosphorylates SMADs and leads to the expression of targets genes.However,the reproductive function of BMPs in invertebrates is undefined,and whether an autocrine/paracrine functional mechanism exists in the ovary of invertebrates,is still unknown.In this paper,we studied the mud crab,Scylla paramamosain,which is an important marine economical aquaculture species in southern China.The full length of the cDNA of SpBMP7,SpBMPRIB and SpBMPRII were obtained via RT-PCR and RACE methods.The nucleotide sequences and deduced amino acid sequences were analyzed and characterized by bioinformatics methods.Quantitative real-time PCR(qRT-PCR),in-situ hybridization,in vitro experiment and dsRNA interference were performed to study the role of SpBMP during ovarian development.In addition,the effects and mechanism of SpBMP7 on oocyte maturation were investigated by separation of oocyte and follicle cells,in vitro experiment and dsRNA interference.The main results are offered as following:1)This study investigated the role of SpBMP during ovarian development.The full length of SpBMP7 cDNA was 1,279 bp with 909 bp open reading frame(ORF)encoding 302 amino acids;the full length of SpBMPRIB cDNA was 2,388 bp with 1,557 bp ORF encoding 518 amino acids;the full length of SpBMPRII cDNA was 2,762 bp with 1,875 bp ORF encoding 624 amino acids.The qRT-PCR data revealed that both ligand and receptors were detected in various tissues,especially in ovary.During the ovarian development,the expression of BMPRs mRNA increased while BMP7 decreased.The results of in-situ hybridization revealed that SpBMP7 was exclusively localized in follicle cells,whereas the receptors(SpBMPRIB and SpBMPRII)were localized in both oocytes and follicle cells.RNAi experiments demonstrated that the mRNA expression levels of Smadl and vitellogenin receptor decrease rapidly after BMPRs were silenced.Based on these data,BMP7 and BMPRs in S.paramamosain were involved in the ovarian development,presumably via the autocrine/paracrine way.2)This study investigated the effects and mechanism of SpBMP7 on oocyte maturation.Oocytes and follicle cells were separated mechanically from mature ovaries.The results of semiquantitative PCR demonstrated that the mRNA of SpBMP7 was exclusively expressed in follicle cells while SpBMPRs were expressed in both oocytes and follicle cells.Further in vitro experiments showed that both mRNA and protein level of cyclin B increased but SpBMP7 declined in 17a,20?-Dihydroxyprogesterone(DHP)-induced oocytes;it was also indicated that the inhibitory effects of SpBMP7 were not influenced by the elimination of the contact/gap junction-mediated communication between oocytes and follicle cells.All these data together hence appear to indicate that BMP7 might suppress DHP-induced oocyte maturation by the way of autocrine/paracrine in S.paramamosain.In conclusion,BMPs have autocrine/paracrine effects on ovarian development and maturation in S.paramamosain and SpBMP7 possiblely inhibit the DHP-induced oocyte maturation.This research might provide new theoretical guidance for the research of reproduction in crustaceans.